Abstract
Opportunistically pathogenic fungi have varying potential to cause disease in animals. Factors contributing to their virulence include specialized metabolites, which in some cases evolved in contexts unrelated to pathogenesis. Specialized metabolites that increase fungal virulence in the model insect Galleria mellonella include the ergot alkaloids fumigaclavine C in Aspergillus fumigatus (syn. Neosartorya fumigata) and lysergic acid α-hydroxyethylamide (LAH) in the entomopathogen Metarhizium brunneum. Three species of Aspergillus recently found to accumulate high concentrations of LAH were investigated for their pathogenic potential in G. mellonella. Aspergillus leporis was most virulent, A. hancockii was intermediate, and A. homomorphus had very little pathogenic potential. Aspergillus leporis and A. hancockii emerged from and sporulated on dead insects, thus completing their asexual life cycles. Inoculation by injection resulted in more lethal infections than did topical inoculation, indicating that A. leporis and A. hancockii were preadapted for insect pathogenesis but lacked an effective means to breach the insect's cuticle. All three species accumulated LAH in infected insects, with A. leporis accumulating the most. Concentrations of LAH in A. leporis were similar to those observed in the entomopathogen M. brunneum. LAH was eliminated from A. leporis through a CRISPR/Cas9-based gene knockout, and the resulting strain had reduced virulence to G. mellonella. The data indicate that A. leporis and A. hancockii have considerable pathogenic potential and that LAH increases the virulence of A. leporis. IMPORTANCE Certain environmental fungi infect animals occasionally or conditionally, whereas others do not. Factors that affect the virulence of these opportunistically pathogenic fungi may have originally evolved to fill some other role for the fungus in its primary environmental niche. Among the factors that may improve the virulence of opportunistic fungi are specialized metabolites--chemicals that are not essential for basic life functions but provide producers with an advantage in particular environments or under specific conditions. Ergot alkaloids are a large family of fungal specialized metabolites that contaminate crops in agriculture and serve as the foundations of numerous pharmaceuticals. Our results show that two ergot alkaloid-producing fungi that were not previously known to be opportunistic pathogens can infect a model insect and that, in at least one of the species, an ergot alkaloid increases the virulence of the fungus.